Overexpression analysis of bilberry (Vaccinium myrtillus L.) R2R3-MYB transcription factors

Abstract

Bilberry (Vaccinium myrtillus L.) is a dwarf shrub and its berries are rich source of nutrients, fibers and vitamins. Bilberry consumption has been shown to positively correlated with improvement of human health. Anthocyanins and proanthocyanidins (PAs) are important secondary metabolites that are synthetized in plants via flavonoid biosynthesis pathway. PAs also have anti-cancer, anti-inflammatory and antioxidant effect in humans. The main aim of this thesis was to identify role of bilberry MYB TFs in biosynthesis of anthocyanin and PAs. Eight R2R3-VmMYB transcription factors (TFs), (VmMYBPA2.1, VmMYBPA2.2, VmMYBPA2.3, VmMYBPA2.4, VmMYBPA2.5, VmMYBPA3, VmMYB5, VmMYB7), were cloned to pGreen-62SK plasmid. PCR amplification verified ligation of genes of interest into pGreen plasmid. Sanger sequencing further confirmed that the full-length sequences were ligated into plasmids. Agroinfiltration was used for overexpression analysis of VmMYBs together with AtbHLH into leaves of Nicotiana benthamiana. Spectrophotometry analysis confirmed that positive control, VmMYBA1 and VmMYBPA2.2 accumulated anthocyanins and PAs. Furthermore, calluses from bilberry fruit explant, were induced in WPM media with hormones, naphthalene acetic acid (NAA), 6-benzylamino purine (BAP), and thidiazuron (TDZ). Appearance of reddish color in calluses after agroinfiltration of VmMYBA1 and VmMYBA2 may indicate the accumulation of anthocyanin in calluses. In this study, it was shown that bilberry VmMYBPA2.2 positively regulates biosynthesis of proanthocyanidins and VmMYBA1 may be a key regulator of biosynthesis of both anthocyanins and proanthocyanidins in Vaccinium myrtillus L.